Use of n-acyl homoserine lactones for the treatment of insulitis

ABSTRACT

An immune response modulatory compound is described. The compound has been shown to inhibit lymphocyte proliferation and to down-regulate TNF-secretion by monocytes and/or macrophages with the consequent activation of Th 1 lymphocytes in humans or other animals.

The invention relates to N-acyl homoserine lactones which haveimmunosuppressant properties and to pharmaceutical compositionscontaining them.

Immunosuppressant compounds induce an inhibition of the immune responsesystem. Compounds which are known to exhibit immunosuppressant activityinclude the fungal metabolite Cyclosporin A and the macrolide antibiotic(a metabolite from Streptomyces tsukabaensis) termed FK506. Both ofthese agents have been used clinically and experimentally to suppressthe immune system in transplantation and in the treatment of a number ofdiseases.

Autoimmune diseases are disorders where the host discrimination of“self” versus “non-self” breaks down and the individual's immune system(both acquired and innate components) attacks self tissues. Thesediseases range from common entities such as rheumatoid arthritis,thyroid autoimmune disease and type 1 diabetes mellitus to less commonentities such as multiple sclerosis and to rarer disorders such asmyasthenia gravis. Advances in basic biomedical science and, inparticular, in immunology have indicated that the main and fundamentallesion responsible for the induction and persistence of most autoimmunediseases resides within auto-reactive proliferating T lymphocytes. Infact, the majority of autoimmune diseases are linked to a loss of T cellhomeostasis. The healthy immune system is held in balanced equilibrium,apparently by the contra-suppressive production of cytokines by T helper1 (Th1) and T helper 2 (Th2) lymphocyte subsets. In autoimmunity, Th1cytokines predominate; in allergy, Th2 cytokines take their place. Acytokine intimately associated with the development of Th1 biasedresponses and, consequently, autoimmune disease is TNF-α.

Both Cyclosporin A and FK506 have been used clinically in the treatmentof autoimmune diseases with encouraging results.

The currently available immunosuppressant drugs have the disadvantage ofa narrow therapeutic index, i.e., toxicity versus clinical benefit. Thecompounds are known to be nephrotoxic, neurotoxic and potentiallydiabetogenic and this has limited their use in the fields mentionedabove. Problems also exist with the administration of these compounds,their bioavailability and the monitoring of their levels both clinicallyand in the laboratory.

We disclosed, in WO-A-95/01175, a class of compounds which exhibitantiallergic activity and inhibit the release of histamine, having thegeneric formula

where: n is 2 or 3; Y is O, S or NH; X is O, S or NH; and R^(a) isC₁-C₁₈ alkyl or acyl which may be substituted.

Some of these compounds, and methods for their preparation, werepreviously disclosed in WO-A-92/18614 although that document disclosesonly that the compounds act as autoinducers and as agents for thecontrol of gene expression. Compounds in the same series are alsomentioned in Journal of Bacteriology, volume 175, number 12, June 1993,pages 3856 to 3862 but again there is no teaching that they might haveany effect outside micro-organisms.

PCT/GB01/01435 describes the use of homoserine lactone compounds fortopical application for autoimmune diseases such as psoriasis. Thepreferred active compound is N-(3-oxododecanoyl)-homoserine lactone. Theactive compound is preferably formulated in an ointment, cream orlotion.

G. Papaccio, Diabetes Res. Clin. Pract. vol.13, no.1, 1991, pages 95-102discloses the use of N-acetylhomocysteine thiolactone as an enhancer ofsuperoxide dismutase in an attempt to increase protection againstchemically induced diabetes.

The use of N-acetylhomocysteine thiolactone to modify the IgE moleculeis taught by J. Ljaljevic et al in Od. Med. Nauka, vol.24, 1971, pages137-143 and Chemical Abstracts, vol.78, no.7, February 1973, abstractno. 41213a.

However, there is no suggestion in this paper of immunosuppression or ofthe inhibition of histamine release.

U.S. Pat. No. 5,591,872 discloses the compound N-(3-oxododecanoyl)homoserine lactone as an autoinducer molecule. In “Infection andImmunity”, vol.66, no.1, January 1998, the authors report the action ofN-(3-oxododecanoyl)homoserine lactone (OdDHL) in inhibiting theconcanavalin A mitogen stimulated proliferation of murine spleen cellsand TNF-α production by LPS-stimulated adherent murine peritonealmacrophages.

We have now discovered a subclass of N-acyl homoserine lactones thatexhibits an immunosuppressant activity greater than that exhibited bysimilar compounds outside of this subclass.

According to one aspect, the present invention provides a compound ofthe formula I

in which R is an acyl group of the formula II

wherein one of R¹ and R² is H and the other is selected from OR⁴, SR⁴and NHR⁴, wherein R⁴ is H or 1-6C alkyl, or R¹ and R² together with thecarbon atom to which they are joined form a keto group, and R³ is astraight or branched chain, saturated or unsaturated aliphatichydrocarbyl group containing from 8 to 11 carbon atoms and is optionallysubstituted by one or more substituent groups selected from halo, 1-6Calkoxy, carboxy, 1-6C alkoxycarbonyl, carbamoyl optionally mono- ordisubstituted at the N atom by 1-6C alkyl and NR⁵R⁶ wherein each of R⁵and R⁶ is selected from H and 1-6C alkyl or R⁵ and R⁶ together with theN atom form a morpholino or piperazino group, or any enantiomer thereof,with the proviso that R is not a 3-oxododecanoyl group.

The compounds of the present invention are capable of modulating theimmune response in the living animal body, including human. Inparticular, they have an inhibitory effect on lymphocyte proliferationin humans and down-regulate TNF-α secretion by monocytes/macrophagesand, in consequence, the activation of Th1 lymphocytes in humans. Thepresent invention, therefore, provides a pharmaceutical compositioncomprising a therapeutically-effective amount of a compound of theinvention as described herein, including an enantiomer thereof, togetherwith a pharmaceutically-acceptable carrier or diluent.

A further aspect of the invention provides the use of a compound of theinvention, including an enantiomer thereof, for the manufacture of amedicament for the treatment of a disease of a living animal bodyincluding human which disease is responsive to the activity of animmunosuppressant, for example an autoimmune disease. A yet furtheraspect of the invention relates to a method of treating a disease of aliving animal body, including a human, which disease is responsive tothe activity of an immunosuppressant, e.g., an autoimmune disease, whichmethod comprises administering to the living animal body, includinghuman, a therapeutically-effective amount of a compound according to theinvention, as described herein including an enantiomer thereof.

Compounds of the invention have the general formula I given above. Thegroup R in the formula I has the formula II

In formula II according to a first preferred embodiment one of R¹ and R²is H and the other is selected from OR⁴, SR⁴ and NHR⁴, in which R⁴ is Hor a 1-6C alkyl group. Preferably, R⁴ is H. Such a definition of R¹ andR² gives rise to chirality at the carbon atom to which R¹ and R² areattached (C-3). The compounds of the invention can, thus, be in the formof racemates, optically active isomers or mixtures thereof. According toa particular preferred embodiment one of R¹ and R² is H and the other isOH.

According to this first preferred embodiment the group R³ in formula IIis a straight or branched chain 8 to 11C aliphatic hydrocarbyl groupwhich is saturated or which may be ethylenically unsaturated. The groupmay, further, be substituted by one or more substituent groups selectedfrom halo, for example F, Cl, Br or I; 1-6C alkoxy, for example methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy and tert-butoxy;carboxy including salts thereof, 1-6C alkoxycarbonyl, for examplemethoxycarbonyl, carbamoyl, for example N,N-dimethylcarbamoyl and NR⁵R⁶,wherein R⁵ and R⁶ are each selected from H and 1-6C alkyl or R⁵ and R⁶together with the nitrogen atom to which they are attached form amorpholino group or a piperazino ring, optionally substituted at the 4-Nby a methyl group. A particularly preferred R³ group in formula II aboveis a straight chain or branched chain 8 to 11C alkyl group which isoptionally substituted by one substituent selected from Br, carboxyincluding salts thereof, and methoxycarbonyl. The substituent istypically, though not necessarily, attached in a terminal position onthe alkyl group. Alternatively, the R³ group is a straight chain orbranched chain 8-11C alkenyl group, preferably monoethenicallyunsaturated, which may be substituted by a substituent selected from Br,carboxy including a salt thereof, and methoxycarbonyl. Again, thesubstituent is typically, though not necessarily, attached in a terminalposition on the alkenyl group.

In formula II above according to a second preferred embodiment thegroups R¹ and R² together form an oxo group (═O) such that a keto groupexists at the C-3 position in the acyl group. In such a case the groupR³ in formula II will typically be:

-   -   (a) an optionally-substituted, saturated or        ethylenically-unsaturated, straight or branched chain 8C        aliphatic hydrocarbyl group;    -   (b) a substituted, saturated, straight or branched chain 9C        aliphatic hydrocarbyl group;        -   10-methyl-3-oxoundecanoyl;        -   6-methyl-3-oxoundecanoyl;        -   3-hydroxydodecanoyl;        -   12-bromo-3-oxododecanoyl;        -   3-oxotridecanoyl;        -   13-bromo-3-oxododecanoyl;        -   3-hydroxytetradecanoyl;        -   3-oxotetradecanoyl;        -   14-bromo-3-oxotetradecanoyl; and        -   13-methoxycarbonyl-3-oxotridecanoyl.

Examples of acyl groups R of formula II above in which R³ is anethylenically unsaturated hydrocarbyl group include:

-   -   3-oxo-12-tridecenoyl;    -   3-oxo-7-tetradecenoyl;    -   3-hydroxy-7-tetradecenoyl;    -   3-oxo-9-tetradecenoyl;    -   3-hydroxy-9-tetradecenoyl;    -   3-oxo-10-tetradecenoyl;    -   3-hydroxy-10-tetradecenoyl;    -   3-oxo-11-tetradecenoyl;    -   3-hydroxy-11-tetradecenoyl;    -   3-oxo-13-tetradecenoyl; and    -   3-hydroxy-13-tetradecenoyl.

The compounds of the present invention having the 3-oxo group may, ingeneral, be prepared by a method comprising the steps of:

-   -   (1) reacting an acid having the general formula R³COOH, where R³        is as defined above, with Meldrum's acid        (2,2-dimethyl-1,3-dioxane-4,6-dione) in the presence of        4-dimethylaminopyridine and N,N¹-dicyclohexylcarbodiimide in a        dry organic solvent, such as dry dichloromethane, to give the        acylated Meldrum's acid; and    -   (c) an optionally-substituted, ethylenically-unsaturated,        straight or branched chain 9C aliphatic hydrocarbyl group;    -   (d) an optionally-substituted, saturated or        ethylenically-unsaturated, straight or branched chain 10C        aliphatic hydrocarbyl group; or    -   (e) an optionally-substituted, saturated or        ethylenically-unsaturated, straight or branched chain 11C        aliphatic hydrocarbyl group.

In the case where the group R³ is substituted, it will be substituted byone or more substituent groups selected from halo, for example F, Cl, Bror I; 1-6C alkoxy, for example methoxy, ethoxy, n-propoxy, iso-propoxy,n-butoxy, iso-butoxy and tert-butoxy; carboxy including salts thereof,1-6C alkoxycarbonyl, for example methoxycarbonyl, carbamoyl, for exampleN,N-dimethylcarbamoyl, and NR⁵R⁶, wherein R⁵ and R⁶ are each selectedfrom H and 1-6C alkyl or R⁵ and R⁶ together with the nitrogen atom towhich they are attached form a morpholino group or a piperazino ring,optionally substituted at the 4-N by a methyl group.

According to one preferred embodiment the R³ group in formula II aboveis a straight chain or branched chain 8, 10 or 11C alkyl group which isoptionally substituted by one substituent selected from Br, carboxyincluding salts thereof, and methoxycarbonyl. According to anotherpreferred embodiment the R³ in formula II above is a straight chain orbranched chain 9C alkyl group which is substituted by one substituentselected from Br, carboxy including salts thereof and methoxycarbonyl.The substituent is typically, though not necessarily, attached in aterminal position on the alkyl group.

According to yet another preferred embodiment the R³ group is a straightchain or branched chain 8-11C alkenyl group, preferably monoethenicallyunsaturated, which may be substituted by a substituent selected from Br,carboxy including a salt thereof, and methoxycarbonyl. The substituentis typically, though not necessarily, attached in a terminal position onthe alkyl group.

Examples of acyl groups R of formula II above in which R³ is a saturatedhydrocarbyl group include:

-   -   3-oxoundecanoyl;    -   11-bromo-3-oxoundecanoyl;    -   (2) reacting the acylated Meldrum's acid with L-homoserine        lactone hydrochloride in an organic solvent, e.g., acetonitrile,        to give the N-(3-oxoacylated)-L-homoserine lactone.

Where the appropriate acid is not available it may be prepared by, forinstance, oxidising the appropriate alcohol using chromic acid.

The N-(3-hydroxyacyl)-L-homoserine lactone may be prepared by reducingthe corresponding N-(3-oxoacyl)-L-homoserine lactone using sodiumcyanoborohydride in acid conditions.

As mentioned above, the compounds of the present invention have use aspharmaceutically active ingredients in the treatment of an animal body,including the human body, suffering from a disease or disorder which isresponsive to the activity of an immunosuppressant, particularly for thetreatment of type I diabetes mellitus (type 1A autoimmune). The dosageadministered to the animal body in need of therapy will, of course,depend on the actual active compound used, the mode of treatment and thetype of treatment desired as well as on the body mass. The activecompound may, of course, be administered on its own or in the form of anappropriate medicinal composition containing, for instance, anappropriate pharmaceutical carrier or diluent. Other substances can, ofcourse, also be employed in such medicinal compositions, such asantioxidants and stabilisers, the use of which is well known to personsskilled in the art.

Preferably, the compound is orally administered. The present inventorshave found that, in the treatment of insulitis or type I diabetesmellitus, at present in NOD (non-obese diabetic) mice, that thecompounds of the present invention have greater efficacy andbioavailability than the conventionally used compound vehicle DMSO.Furthermore, the compounds of the present invention perform at leastcomparably to accepted immune modulators such as CsA or antibodies toTNF without exerting overt immune toxicity.

In comparative in vitro studies for the prevention of cell proliferationCSA and dexamethasone performed better than OdDHL. However,unexpectedly, when the assay was performed in vivo OdDHL performedbetter than CSA and dexamethasone (see below), leading the presentinventors to conclude that in an in vivo situation CSA and dexamethasoneare either inactivated or prevented from acting by metabolic processesor by physiological breakdown of the compound whereas OdDHL is eitherresistant to these processes or changes or is somehow further activatedby them. From this it is believed that a similar result will be achievedfor insulitis.

The most preferred compound of the present invention isOdDHL[N-(3-oxododecanoyl)-L-homoserine lactone] and derivatives orsubstituents thereof, as set out in PCT/GB01/01453, the content of whichis incorporated herein by reference.

In another aspect of the invention, the OdDHL or the other relatedimmune modulatory compounds may be used in the identification ofmolecular targets and novel immunophilins in cells, preferablypancreatic beta cells or autoreactive leucocytes by constructingaffinity matrices incorporating the compounds.

Two possible chemical strategies which may be employed in the productionof such an affinity matrix are the synthesis of side-chainfunctionalised, for example C2 or C4-functionalised OdDHL (3O, C12-HSL)derivates (X=(CH₂)_(n)COOH) as shown for side chains in Formulae I andII below. The length of the alkyl chain (n=1, 2, 3 etc) will determinethe efficiency of binding to the novel immunophilins.

In the above formulae, X may be selected from Br, Cl, I or (CH₂)nCOOH.

Alternatively, the molecule may be terminally functionalised followingthe schemes shown in FIGS. 13 and 14.

In a further aspect of the invention, more potent immune modulatoryagents can be generated by the synthesis of bivalent OdDHL, PQS andhybrids. These bivalent ligands can be constructed by, linking PQS andHdDHL (3OH, C12-HSL) through a spacer of optimum length to provide aHomo-dimer (See FIG. 1) or Hetero-dimer (See FIG. 2).

Preferably, the two molecules will be linked though their respective3-OH substituents via a spacer. Alternatively, for the Homo-dimers, whenthe C2 or C4 substituent is carboxyalkyl [(CH_(2)n)COOH], OdDHL-dimerscan be synthesised by the method described in J.Med. Chem. 2001, 44,1615-1622. Without wising to be bound by theory, the present inventorsbelieve that the nature of the substituent at C2 or C4, that is thehalogen or the carboxyalkyl will determine or limit the nature of thespacer linking the two molecules. For example, the choice of the spaceris likely to have an effect on the immunomodulatory properties of thedimer through possible sites of the linkage of the spacer and the natureof the covalent linkage. Additionally, the length of the spacer isimportant when determining the effectiveness of the cross-linking sinceit is desired to create a compound which exhibits a potency that isgreater than that derived from the sum of its two monovalentpharmacophores. It is desirable to use a flexible or conformationallyrestricted spacer, especially in order to prevent adverse effect withsteric hindrance. Some examples of spacers are shown in FIG. 3.

Optionally, the carboxyalkyl substituent at C2 or C4 can be tagged (forexample, with a colorimetric or fluorometric tag) for cellcompartmentalisation assays or targeting studies, or simply for use inthe affinity matrix studies, for example to show binding. This strategyof tagging via C2 or C4 is especially preferred as the biologicallyimportant parts of the molecule are unaffected or at least are stillavailable for chemical, biochemical or physiological interactions.

Embodiments of the invention will now be described in more detail, byway of example only, with reference to the following examples which areillustrated with reference to the accompanying drawings, of which

FIGS. 1, 2 and 3 have already been described;

FIG. 4 is a graph showing the comparative in vitro anti proliferativeeffects of CSA, dexamethasone and OdDHL;

FIG. 5 is a graph showing the influence of oral OdDHL on a murine invivo DTH response to SRBCs;

FIG. 6 is a graph showing the proliferation of Balb/C splenocytesstimulated with ConA. in the presence of OdDHL and OOHL;

FIG. 7 is a graph showing the proliferation of human PBMC stimulatedwith ConA in the presence of OdDHL and OtDHL;

FIG. 8 is a graph showing TNFα production by human PBMC stimulated withLPS in the presence of OdDHL and OtDHL;

FIG. 9 is a graph showing TNF-α production in the presence of testcompounds;

FIG. 10 is a graph showing TNF-α production by human PBMC in thepresence of test compounds;

FIG. 11 is a graph showing TNFα production in the presence of drugs;

FIG. 12 is a graph showing lack of overt toxicity of OdDHL.

FIG. 13 shows schematically the synthesis of a terminally functionalisedOdDHL; and

FIG. 14 shows schematically the synthesis of a terminally functionalisedOdDHL.

EXAMPLES Example 1

N-(3-oxododecanoyl)-L-homoserine Lactone (OdDHL)

To a solution of decanoic acid (2 mmol) in dry dichloromethane (20 ml)was added 4-dimethylaminopyridine (2.1 mmol),N,N′-dicyclohexylcarbodiimide (2.2 mmol) and Meldrum's acid (2 mmol).The solution was stirred at room temperature overnight and then filteredto remove the precipitated dicyclohexylurea. The filtrate was evaporatedto dryness and the residue redissolved in ethyl acetate. The ethylacetate solution was washed with 2 M hydrochloric acid, dried overanhydrous magnesium sulphate and concentrated to afford the decanoylMeldrum's acid.

To a stirred solution of the decanoyl Meldrum's acid (1 mmol) inacetonitrile (30 ml) was added L-homoserine lactone hydrochloride (1mmol) and triethylamine (1.2 mmol). The mixture was stirred for 2 hoursand then refluxed for a further 3 hours. The solvent was removed byrotary evaporation to give a residue that was redissolved in ethylacetate. The organic solution was sequentially washed with saturatedsodium hydrogen carbonate solution, 1M potassium hydrogen sulphatesolution and saturated sodium chloride solution. After drying overanhydrous magnesium sulphate, the organic extract was evaporated todryness and the residue purified by preparative layer chromatography onsilica plates.

¹H NMR (250 MHz, CDCl₃) δ 0.9 (3H, t, CH₃), 1.27 (12H, m, CH₃(CH₂)₆),1.59 (2H, m, CH₂CH₂CO), 2.22 (1H, m, 4α-H), 2.52 (2H, t, CH₂CO), 2.76(1H, m, 4β-H), 3.47 (2H, s, COCH₂CO), 4.27 (1H, m, 5α-H), 4.48 (1H, td,5β-H), 4.58 (1H, m, 3-H), 7.64 (1H, d, NH).

The procedure described above in Example 1 was followed to prepare otherN-(3-oxoacylated)-L-homoserine lactones as described below using, ineach case, the appropriate carboxylic acid.

Example 2

N-(12-Bromo-3-oxododecanoyl)-L-homoserine Lactone (12BrOdDHL)

¹H NMR (250 MHz, CDCl₃) δ 1.27 (10H, m, BrCH₂CH₂(CH₂)₅), 1.45 (2H, m,BrCH₂CH₂CH₂), 1.59 (2H, m, CH₂CH₂CO), 2.22 (1H, m, 4α-H), 2.52 (2H, t,CH₂CO), 2.76 (1H, m, 4β-H), 3.47 (2H, s, COCH₂CO), 3.53 (2H, t, BrCH₂),4.27 (1H, m, 5α-H), 4.48 (1H, td, 5β-H), 4.58 (1H, m, 3-H), 7.64 (1H, d,NH).

Example 3

N-(12-Hydroxy-3-oxododecanoyl)-L-homoserine Lactone (12OHOdDHL)

Using 10-acetoxydecanoic acid in the general procedure as describedabove in Example 1 afforded theN-(12-acetoxy-3-oxododecanoyl)-L-homoserine lactone. The latter whenrefluxed in 1M hydrochloric acid, yielded the title product.

¹H NMR (250 MHz, CDCl₃) δ 1.27 (12H, m, HOCH₂(CH₂)₆), 1.59 (2H, m,CH₂CH₂CO), 1.89 (1H, t, OH), 2.22 (1H, m, 4α-H), 2.52 (2H, t, CH₂CO),2.76 (1H, m, 4β-H), 3.47 (2H, s, COCH₂CO), 3.60 (2H, t, HOCH₂), 4.27(1H, m, 5α-H), 4.48 (1H, td, 5β-H), 4.58 (1H, m, 3-H), 7.64 (1H, d, NH).

Example 4

Synthesis of a Homo and a Hybrid-dimer by Linking through 3-OHSubstituents

N,N′-Dicyclohexylcarbodiimide (DCCl) and 4-dimethylaminopyridine (DMAP)catalysed acylation of the 3-OH substituent of the PQS with glutaricacid mono t-butyl ester would furnish the acylated PQS (Scheme 1).Removal of the tBu protection by acidolysis is followed byesterification of the resultant carboxylic acid either with the 3-OHsubstituent of another molecule of PQS to furnish the homo-dimer orHdDHL (3-OH, C12-HSL) to deliver the desired hetero-dimer.

Example 5

Effect of OdDHL on Insulitis

To investigate the effect of OdDHL on insulitis or type I diabetesmellitus, mice (NOD) with a genetic predisposition to develop IDDM(insulin dependent diabetes mellitus) were treated with OdDHL.

The animals were dosed at 100 mg/kg intraperitoneally from 4 weeks ofage, 3 times a week for four weeks. Insulitis was assessed at 14 weeks.

In the table a score of 3 or more (DMSO and PBS) represents severe gradeinsulitis and 1.3 (OdDHL) represents little infiltration (a score of 1would represent low grade peri-insulitis).

The results for 100 mg/kg are shown in Table 1 below. The mice werescored according to the method of Beales et al (European Journal ofPharmacology 357(1998) 221-225). TABLE 1 DMSO PBS OdDHL Animal NumberSlide Score Slide Score Slide Score  1 3.160  2 1.000  3 1.418  4 3.372 5 1.891  6 3.855  7 4.352  8 3.937  9 1.936 10 3.202 11 1.478 12 2.69013 3.806 14 1.735 15 3.184 16 1.154 17 2.019 Group Total 18.23 18.8806.986 Group Mean 3.054 3.140 1.397 Standard 1.01691 0.76115 0.35853Derivation

Example 6

Oral Availability of OdDHL

To show that OdDHL is orally available, the anti-sheep red blood cell(SRBC) responses of mice were measured in accordance with the followingexperiment. Eight week old, female Balb/c mice were kept on a 12 hourlight/dark cycle and fed food and water ad libitum. SRBCs in Alsevierssolution (TCS Biologicals, SB069) were washed and centrifuged (800×g for20 minutes at room temperature) three times with 0.9% NaCl in distilledwater. Cells were counted using a haemocytometer. On day 0 animals wereimmunised intraperitoneally (IP) with 5×10⁶ (low dose) or 5×10⁸ (highdose) SRBCs in saline. On day 5, animals were challenged with 20 μlSRBCs at 5×10⁹/ml in the left hind paw. The contralateral paw wasinjected with 20 μl saline alone. After 24 hours the animals weresacrificed with a rising concentration of CO₂ and bled by cardiacpuncture. The hind feet were severed at the ankle joint and weighed.

Animals were dosed po from day 0 to day 5 with OdDHL. OdDHL wassuspended in 0.25% (wt/vol) cellosize (Boots Co. PLC, Nottingham UK) indistilled water containing 1.5% (vol/vol) Tween 80 (Registered TradeMark, Sigma) for po dosing at 3, 10, 30 and 100 mg/kg. Animals received0.1 ml po.

It was found that immunisation of Balb/c mice with 5×10⁶ SRBCs evokes adelayed type hypersensitivity response (type IV) when challenged withSRBCs in the footpad. This manifests itself as an inflammatory responsecausing an increase in the size of the foot. Very little antibody isproduced. OdDHL at 3, 10, 30 and 100 mg/kg caused a dose dependentincrease in footweight of 37%, 60%, 100% and 93% respectively (see FIG.5). Mice dosed with OdDHL had higher footweights when compared tovehicle dosed animals. The 30 and 100 mg/kg doses (** on graph) aresignificant as determined by Dunnett's multiple comparison's test afterone-way analysis of variance (p<0.01). This assay is considered by thepharmaceutical industry to be Th2 dependent, indicating the effect ofcompound treatment on contra-regulating T-helper 1 lymphocytes.

Example 7

Immunomodulatory Activity of Homoserine Lactone Compounds

Materials and Methods

I. ConA Mitogen-stimulated Proliferation of Murine Splenocytes

The concanavalin A (ConA) cell proliferation assay was used to assessthe effect of homoserine lactone (HSL) compounds on T-cell activationand proliferation. Proliferation was assessed by the incorporation of[³H]-thymidine into DNA. Eight-week-old female BALB/c mice were obtainedfrom Harlan (Bicester, Oxon, UK) and given food and water ad libitum.Splenocyte suspensions were prepared by removing the spleens and placingthem into. RPMI 1640 medium. The spleens were forced through70-μm-pore-size wire gauzes using the plunger from a 5-ml syringe toproduce a single cell suspension. The cells were pelleted bycentrifugation, and erythrocytes were lysed with 0.017M Tris, 0.144Mammonium chloride buffer, pH 7.2. Leucocytes were washed twice with RPMI1640 medium with 2% (vol/vol) foetal calf serum (FCS) and resuspended incomplete cell culture medium (CTCM) consisting of RPMI 1640 medium with5% FCS, 2 mM L-glutamine, and 5×10⁻⁵ M 2-mercaptoethanol. HSL compoundswere tested at doubling down dilutions ranging from 1 mM to 0.1 μM in afinal volume of 200 μl of CTCM, containing ConA (Sigma, Poole, UK) at 1μg/ml and 100,000 spleen cells. Following incubation for 48 h at 37° C.in 5% CO₂-air, 0.25 μCi [³H]-thymidine (Amersham) in 10 μl volume madeup in RPMI 1640 medium was added and the cells were incubated for afurther 24 h. Cells were harvested onto fibreglass filters with aPackard filtermate harvester. After the addition of 25 μl ofMicroScint-O (Packard) to each well the filters were counted with thePackard TopCount scintillation counter.

Mitogen (Concanavalin A) induced murine splenocyte proliferation wasindicated by the incorporation of tritated thymidine into the DNA in themouse spleen cells as shown by counts per minute using the scintillationcounter. The inhibitory effect of an HSL compound being tested on cellproliferation was indicated by a reduction in counts per minute. FIG. 6shows the plots of counts per minute (cpm) against the concentrations(micromolar) of the HSL compounds N-(3-oxododecanoyl)-L-homoserinelactone (OdDHL) and N-(3-oxooctanoyl)-L-homoserine lactone (OOHL) andthe vehicle dimethylsulphoxide (DMSO). It can be seen, from this figure,that OdDHL inhibits splenocyte proliferation. In contrast, OOHL and DMSOfailed to inhibit proliferation.

The IC50 value, i.e., the concentration (micromolar) of a compound whichinhibits cell proliferation thymidine incorporation by 50% wasdetermined for several compounds of the present invention and these IC50 values are shown in column A of the Table below.

II. ConA Mitogen-stimulated Proliferation of Human PBMC

Blood specimens were obtained with consent from healthy humanvolunteers. Human peripheral blood mononuclear cells (PBMC) wereisolated from heparinised whole blood by buoyant density centrifugationover Histopaque 1077 (Sigma, Poole, UK) at 600 g for 20 minutes. PBMCharvested from the ‘buffy’ layers were washed twice with RPMI 1640medium and resuspended in CTCM. HSL compounds were tested at similardilutions as for murine splenocytes in 200 μl of CTCM, containing 1μg/ml of ConA and 100,000 PBMC. Human PBMC were incubated for 48 h at37° C. in 5% CO₂-air, followed by pulsing with 0.25 μCi [³H]-thymidine(see above). After a further incubation of 24 h cells were harvestedonto fibreglass filters and then counted in the presence of MicroScint-Owith the Packard TopCount.

Concanavalin induced cell proliferation of human peripheral bloodmononuclear cells (PBMC) was tracked, as described in I above, by ameasurement of counts per minute using the scintillation counter. Theinhibitory effect of an HSL compound being tested on cell proliferationwas indicated by a reduction in counts per minute. FIG. 7 shows theplots of cpm against the concentrations of OdDHL,N-(3-oxotetradecanoyl)-L-homoserine lactone (OtDHL) and DMSO (vehicle).As can be seen, both OdDHL and OtDHL inhibited proliferation of humanPBMC stimulated with Concanavalin A.

The IC50 values for several HSL compounds of the invention weredetermined and these are shown in columns B, C and D in the Table below.Columns B, C and D represent different sources of human PBMC samplesused.

III. TNF-alpha Production from LPS-stimulated Human PBMC

Bacterial lipopolysaccharide (LPS) stimulates the production of avariety of cytokines, including TNF-alpha, from human PBMC; thesecytokines in turn influence the development of T cells, supporting a Thelper 1 conducive milieu. Human PBMC prepared from whole blood bybuoyant density centrifugation were resuspended in CTCM. HSL compoundswere again tested at similar dilutions as for murine splenocytes in 200μl of CTCM, containing 5×10⁻⁵ μg/ml LPS Escherichia coli strain 055:B5(Sigma, Poole, UK) and 100,000 PBMC. Following incubation for 24 h at37° C. in 5% CO₂-air, the cell culture supernatants were collected andtested for TNF-alpha levels by ‘sandwich’ ELISA. Briefly, 96-well NuncMaxiSorp (Life Technologies, Paisley, UK) plates were coated with 50 μlof a 2 μg/ml solution of mouse anti-human TNF-alpha monoclonal antibody(Pharmingen, UK) in 0.05 M carbonate/bicarbonate buffer, pH 9.6overnight at 4° C. After washing the plates three times with PBS-Tween,which contained phosphate buffered saline (PBS) with 0.5% (vol/vol)Tween 20 (Sigma, Poole, UK), the plates were blocked with 1% (wt/vol)bovine serum albumin (BSA) (Sigma, Poole, UK) at room temperature for 2h. Following three washes with PBS-Tween, 50 μl of cell culturesupernatants were added and incubated overnight at 4° C.; standard humanTNF-alpha (Pharmingen, UK) ranging from 2000 to 31.25 pg/ml wereincluded for each plate. After four washes with PBS-Tween, 50 μl of asecond antibody, biotinylated mouse anti-human TNF-alpha monoclonalantibody (Pharmingen, UK) was added at 0.5 μg/ml diluted in 1% BSA inPBS-Tween and incubated at room temperature for 1 h. Following fourwashes, the bound biotinylated antibody was detected with 50 μl of a1:1,000 dilution of Streptavidin-peroxidase (Pharmingen, UK). At the endof an hour incubation at room temperature, the plates were thoroughlywashed six times with PBS-Tween and the assay was developed by theaddition of 100 μl of 0.1 mg/ml of tetramethyl benzidine subtrate(Sigma, Poole, UK) in 0.1 M sodium acetate buffer, pH 6 containing 0.03%H₂O₂. The enzyme reaction was stopped with 50 μl of 2.5 M H₂SO₄ after anincubation of 10 minutes at room temperature and the development wasread at 450 nm with a spectrophotometric 96-well plate reader (Dynex).

The effect of the concentration of certain HSL compounds of theinvention on LPS induced TNF-α production by human PBMC was observed.FIG. 8 shows plots of TNF-α concentrations (pg/ml) against theconcentration (micromolar) of OdDHL, OtDHL and DMSO (vehicle). As can beseen, both OdDHL and OtDHL inhibited the secretion of the T helper1-supporting cytokine TNF-α. The IC50 values, i.e., the concentration(micromolar) of a compound which inhibits TNF-α secretion by 50%, wasdetermined for some of the HSL compounds of the invention and these areshown in column E in the Table below.

Similar studies were carried out using, as the HSL compounds,N-(12-bromo-3-oxododecanoyl)-L-homoserine lactone (12BrOdDHL) andN-(12-hydroxy-3-oxododecanoyl)-L-homoserine lactone (12hydroxyOdDHL) andthe plots for these are shown in FIG. 9. For comparison purposes,similar studies were carried out using, as the HSL, the known shorterside chain compound N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) andthe plot for this is shown in FIG. 10. The difference in activitybetween OHHL and OdDHL is marked. Also for comparison purposes, similarstudies were carried out using the known drugs dexamethasone andCyclosporin A (CsA) and the plots for these are shown in FIG. 6. TheIC50 value for dexamethasone was determined to be 500.

IV. Optimisation of Cell Culture Conditions

In the cell culture assays the number of cells used (mouse splenocytesand human PBMC) was initially optimised to 100,000 cells per well. Theoptimal dose of ConA of 1 μg/ml used in the cell proliferation assayswas determined from ConA titration curves. A similar titration curve wasestablished for LPS stimulation to obtain an LPS concentration whichstimulated a suboptimal level of TNF-alpha release from human PBMC.

Example 8

Comparative Immune Toxicity Assessment

The overt immune toxicity of OdDHL was investigated using the doseregime described above in Example 5 for the alleviation of insulitis ordiabetes in NOD mice. Treated mice gained weight identically to theirnon-treated littermates. The results are shown in FIG. 12. Splenocytesfrom the mice were taken along the time course of treatment (3 times aweek for 4 weeks) and were stained with anti-CD-3 (pan T-cell), anti-CD4(helper T-cell), anti-CD8 (cytotoxic T-cell) and anti-CD19 (B cell)antibodies for 30 minutes on ice. Cells were washed twice in PBS/BSA andthen fixed in 0.5% formaldehyde. Cell phenotypes were analysed on BDFACScan. As can be seen from FIG. 12, a lack of overt immune toxicologywas displayed during treatment with the immune cell populationsremaining constant in proportion throughout the course of treatment.

Example 9

In vivo Effects of CSA. Anti-TNFα and OdDHL

Dose regimes of Cyclosporin A, anti-TNFα antibody and OdDHL werecompared in the diabetes model. NOD mice were dosed at 25 mg/kg onalternate days for 160 days following the methodology of Mori et al(Diabetologia (1986) 29: 244-247), the content of which is incorporatedherein by reference, for the investigation of CSA, and Anti TNF at 12 mgper individual, 0.5 mg three times a week for 8 weeks in accordance withthe method of Suk et al (J. Immunology (2001) 166: 4481-4489), thecontent of which is incorporated herein by reference, was used for TNFα.OdDHL was dosed at 30 mg/kg, 3 times per week for 4 weeks as above. Whencompared to the reported data of Mori and Suk (Supra) OdDHL displayedmore effective in vivo action than in vitro contrary to what would beexpected from the in vitro results previously discussed (page 8).

Example 10

Confirmation of Alleviation of Diabetes in NOD Mice

The ability of the lead compound (OdDHL) to alleviate diabetes in NODmice was determined by treating the mice 3 times per week for 4 weeks at30 mg/kg as above. DMSO and OHHL were used as controls.

Diabetes was diagnosed using Uristix and a level of above 6 mMolglucose/I was taken to indicate the presence of diabetes. The resultsare shown in Table 2, which shows the incidence of diabetes atsignificant weeks of the experiment. TABLE 2 Week of Experiment 20 30DMSO 7/12 15/20 OHHL 7/10 12/20 OdDHL 0/11  5/20

A Kaplan-Meier analysis of cumulative incidence of diabetes shows: OdDHLvs DMSO p = 0.0004 OdDHL vs OHHL p = 0.009 DMSO vs OHHL   = nonsignificant (NS)

1. An immune response modulatory compound of the formula I

in which R is an acyl group of the formula II

wherein one of R¹ and R² is H and the other is selected from OR⁴, SR⁴and NHR⁴, wherein R⁴ is H or 1-6C alkyl, or R¹ and R² together with thecarbon atom to which they are joined form a keto group, and R³ is astraight or branched chain, saturated or unsaturated aliphatichydrocarbyl group containing from 8 to 11 carbon atoms and is optionallysubstituted by one or more substituent groups selected from halo, 1-6Calkoxy, carboxy, 1-6C alkoxycarbonyl, carbamoyl optionally mono- ordisubstituted at the N atom by 1-6C alkyl and NR⁵R⁶ wherein each of R⁵and R⁶ is selected from H and 1-6C alkyl or R⁵ and R⁶ together with theN atom form a morpholino or piperazino group, or any enantiomer thereof,with the proviso that R is not a 3-oxododecanoyl group.
 2. A compoundaccording to claim 1, in which group R in the formula I has the formulaII

in which one of R¹ and R² is H and the other is selected from OR⁴, SR⁴and NHR⁴, in which R⁴ is H or a 1-6C alkyl group.
 3. A compoundaccording to claim 2, in which R⁴ is H.
 4. A compound according to claim3, in which one of R¹ and R² is H and the other is OH.
 5. A compoundaccording to any one of claims 1 to 4, in which R³ of formula II is astraight or branched chain 8 to 11C aliphatic hydrocarbyl group which issaturated or ethylenically unsaturated.
 6. A compound according to claim5, in which the R³ group may be further substituted by one or moresubstituent groups selected from halo, F, Cl, Br or I; 1-6C alkoxy,methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy andtert-butoxy; carboxy including salts thereof, 1-6C alkoxycarbonyl,methoxycarbonyl, carbamoyl, N,N-dimethylcarbamoyl and NR⁵R⁶, wherein R⁵and R⁶ are each selected from H and 1-6C alkyl or R⁵ and R⁶ togetherwith the nitrogen atom to which they are attached form a morpholinogroup or a piperazino ring, optionally substituted at the 4-N by amethyl group.
 7. A compound according to claim 5, in which R³ group informula II is a straight chain or branched chain 8 to 11C alkyl groupwhich is optionally substituted by one substituent selected from Br,carboxy including salts thereof, and methoxycarbonyl.
 8. A compoundaccording to claim 5, in which R³ group in formula II is a straightchain or branched chain 8-11C alkenyl group, preferably monoethenicallyunsaturated, which may be substituted by a substituent selected from Br,carboxy including a salt thereof, and methoxycarbonyl.
 9. A compoundaccording to any one of claims 1 to 8, in which the groups R¹ and R²together form an oxo group (═O) such that a keto group exists at the C-3position in the acyl group.
 10. A compound according to claim 9, inwhich the group R³ in formula II will typically be: (a) anoptionally-substituted, saturated or ethylenically-unsaturated, straightor branched chain 8C aliphatic hydrocarbyl group; (b) a substituted,saturated, straight or branched chain 9C aliphatic hydrocarbyl group;(c) an optionally-substituted, ethylenically-unsaturated, straight orbranched chain 9C aliphatic hydrocarbyl group; (d) anoptionally-substituted, saturated or ethylenically-unsaturated, straightor branched chain 10C aliphatic hydrocarbyl group; or (e) anoptionally-substituted, saturated or ethylenically-unsaturated, straightor branched chain 11C aliphatic hydrocarbyl group, and in the case wherethe group R³ is substituted, it is substituted by one or moresubstituent groups selected from the group consisting of halo, F, Cl, Bror I; 1-6C alkoxy, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy,iso-butoxy and tert-butoxy; carboxy including salts thereof, 1-6Calkoxycarbonyl, methoxycarbonyl, carbamoyl, N,N-dimethylcarbamoyl, andNR⁵R⁶, wherein R⁵ and R⁶ are each selected from H and 1-6C alkyl or R⁵and R⁶ together with the nitrogen atom to which they are attached form amorpholino group or a piperazino ring, optionally substituted at the 4-Nby a methyl group.
 11. A compound according to claim 7 or claim 8, inwhich the substituent is attached in a terminal position on the alkylgroup.
 12. A compound according to any preceding claim, in which theacyl groups R of formula II in which R³ is a saturated hydrocarbyl groupinclude: 3-oxoundecanoyl; 11-bromo-3-oxoundecanoyl;10-methyl-3-oxoundecanoyl; 6-methyl-3-oxoundecanoyl;3-hydroxydodecanoyl; 12-bromo-3-oxododecanoyl; 3-oxotridecanoyl;13-bromo-3-oxododecanoyl; 3-hydroxytetradecanoyl; 3-oxotetradecanoyl;14-bromo-3-oxotetradecanoyl; and 13-methoxycarbonyl-3-oxotridecanoyl.13. A compound according to any preceding claim, in which the acylgroups R of formula II in which R³ is an ethylenically unsaturatedhydrocarbyl group include: 3-oxo-12-tridecenoyl; 3-oxo-7-tetradecenoyl;3-hydroxy-7-tetradecenoyl; 3-oxo-9-tetradecenoyl;3-hydroxy-9-tetradecenoyl; 3-oxo-10-tetradecenoyl;3-hydroxy-10-tetradecenoyl; 3-oxo-11-tetradecenoyl;3-hydroxy-11-tetradecenoyl; 3-oxo-13-tetradecenoyl; and3-hydroxy-13-tetradecenoyl.
 14. Use of a compound according to any oneof claims 1 to 13 in a medicament for the modulation of immune responsein the animal body.
 15. Use of a compound according any one of claims 1to 13, in which the animal is a mammal.
 16. Use of a compound accordingto any one of claims 1 to 13, in which the mammal is a human.
 17. Use ofthe compound of any one of claims 1 to 13 in the manufacture of amedicament for the inhibition of lymphocyte proliferation.
 18. Use ofthe compound of any one of claims 1 to 13 in the manufacture of amedicament for the down-regulation of TNF-□ secretion bymonocytes/macrophages and the consequent activation of Th 1 lymphocytesin humans.
 19. A pharmaceutical composition comprising atherapeutically-effective amount of the compound according to any one ofclaims 1 to 13 or an enantiomer thereof.
 20. Use of the compound of anyone of claims 1 to 13 including enantiomers thereof, for the manufactureof a medicament for the treatment of a disease of a living animal bodyincluding human which disease is responsive to the activity of animmunosuppressant.
 21. Use according to claim 20, in which the diseaseis an autoimmune disease.
 22. A method of treating a disease of a livinganimal body, including a human, which disease is responsive to theactivity of an immunosuppressant, e.g., an autoimmune disease, whichmethod comprises administering to the living animal body, includinghuman, a therapeutically-effective amount of a compound according toclaim
 1. 23. Use of compound according to any one of claims 1 to 13, inwhich the compound is orally administered.